RU2751331C1 - Medicinal product for treatment of cerebral circulation impairments and regeneration of lost brain function - Google Patents

Abstract

FIELD: pharmaceuticals.SUBSTANCE: invention relates to the area of pharmacology, namely to a protein-peptide complex exhibiting neuroprotective and neuroregenerative properties. A protein-peptide complex exhibiting neuroprotective and neuroregenerative properties including a mixture of protein molecules and peptides with a molecular weight of 2500 to 200000 Daltons, obtained from a brain of a pig subjected to intravital damage to brain tissue by mechanical action and isolated by homogenisation of the animal brain tissue, purification of the homogenate from solid components resulting in a supernatant, purification of the obtained supernatant from lipid components resulting in an unclarified protein-peptide complex, isolation of a mixture of protein molecules and peptides with a molecular weight of 2500 to 200000 Daltons from the obtained unclarified protein-peptide complex resulting in a protein-peptide complex.EFFECT: described complex has increased efficiency in treatment of both ischemic and hemorrhagic strokes and is technically easier in production.3 cl, 8 tbl, 2 ex

Description

The invention relates to the field of pharmacology, neurology, including children.

The problem of cerebrovascular diseases is of high importance in many countries of the world. Stroke is the leading cause of disability in the population; the likelihood of its development in patients with arterial hypertension increases 3-4 times. About 10 million cases of stroke are diagnosed in the world every year, of which more than 450 thousand are in Russia. Stroke mortality in Russia is 1.23 per 1000 population; within a year after a stroke, about 50% of patients die. Ischemic strokes account for 70-85% of the total number of strokes, intracerebral hemorrhages - 20-25%, non-traumatic subarachnoid hemorrhages - 5%. There are no reliable data on the number of patients with chronic cerebrovascular accidents in Russia, however, according to estimates, the incidence of chronic cerebral ischemia exceeds 700 per 100,000 population (https://ru.wikipedia.org/wiki/Cerebrovascular_diseases). The development of new effective drugs for the treatment of cerebrovascular diseases is an important scientific and technical problem.

From the prior art known drug "Cerebral" for the treatment of stroke (RF Patent 2151605), which is an amino acid - peptide complex containing peptides with a molecular weight of up to 1500 Da and free amino acids. The disadvantage of this invention is the low efficiency in the treatment of ischemic stroke (RF patent 2470650).

The closest analogue of the claimed invention is a drug for the treatment of ischemic and hemorrhagic stroke, isolated from the brain of pigs subjected to modeling of hemorrhagic stroke (patent RU 2470650). However, this drug is not effective enough and, at the same time, difficult to obtain. In particular, to obtain the specified drug, it is necessary to simulate hemorrhagic hemispheric stroke after occlusion of the common carotid artery on the same side (ischemic stroke)

Thus, the object of the claimed invention is to provide a drug for the treatment of cerebrovascular accidents and restoration of lost brain functions, which is effective in the treatment of both ischemic and hemorrhagic stroke and, at the same time, is technically easy to obtain.

The solution to this problem is provided by a protein-peptide complex with neuroprojector and neuroregenerative properties, characterized in that it contains a mixture of protein molecules and peptides with a molecular weight of 2500-200000 Daltons, isolated from the brain of an animal subjected to intravital brain damage by mechanical action.

The authors have shown that after damage to the brain tissue, the content of such proteins / peptides as, for example, Complexin-1, Heat shock 70 kDa protein, Peroxiredoxin-6, Actin, Prothymosin alpha, S100-A6, Parathymosin, S- phase kinase-associated protein 1, Transaldolase, Carbonic anhydrase 2, Neurofilament light polypeptide, Tubulin alpha-4A chain, Tubulin alpha-1D chain, Tubulin beta-2B chain, Microtubule-associated proteins 1A / 1B light chain 3A, ADP-ribosylation factor 1, Tubulin beta-3 chain, Alpha-synuclein, Beta-synuclein, S100-A1, Cystatin-B, Macrophage migration inhibitory factor, Heat shock 90-alpha protein, Phosphatidylethanolamine-binding protein 1 (precursor of Hippocampal cholinergic neurostimulating peptide), Rab GDP dissociation inhibitor alpha, Alpha-enolase, Fatty acid-binding protein (brain), Thioredoxin, 14-3-3, Calmodulin, L-lactate dehydrogenase B chain, Phosphoglycerate kinase 1, Triosephosphate isomerase, Dihydropyrimidinase-related protei n 2, Purine nucleoside phosphorylase, Ras-related proteins Rab-11A, 1A, 3A, 3C, 7a. However, there is reason to believe that not all proteins, the content of which increases in the brain after brain damage, are involved in the mechanisms of neuroprotection and neuroregeneration.

Presumably, protein molecules and peptides, the synthesis of which selectively increases after damage to the brain tissue of an animal, and at the same time, participating in the mechanisms of neuroprotection and neuroregeneration (and, therefore, effective in the treatment of cerebrovascular diseases) have a molecular weight of at least 2500 and at most 200000 Dalton.

One of the optimal combinations of these proteins and peptides, representing the claimed drug, may be the following: Complexin-1; Heat shock 70 kDa protein; 14-3-3; Alpha-synuclein, Beta-synuclein; Fatty acid-binding protein, brain; Phosphatidylethanolamine-binding protein 1 (or Hippocampal cholinergic neurostimulating peptide, of which it is a precursor), Dihydropyrimidinase-related protein 2, Cystatin-B, Tenascin, Cell division cycle 5-like protein, Macrophage migration inhibitory factor.

The composition of the protein-peptide complex indicated in Table 1 determines its specificity and activity.

Additionally, proteins and peptides with neuroprotective properties, such as Aspartate aminotransferase, cytoplasmic, can be added to the preparation; Glucose-6-phosphate isomerase, Protein DJ-1 (Table 2).

In this case, damage to the brain tissue of an animal can be carried out both by mechanical action.

As animals, mammals, for example pigs, can preferably be used.

To a large extent, the solution to this problem is provided by a method for obtaining the above protein-peptide complex, which includes:

- damaging mechanical effect on the brain tissue of the animal;

- fragmentation of animal brain tissue to obtain fragmented parts of animal brain tissue;

- homogenization of the fragmented parts of the animal brain tissue at pH 7.3-7.6 to obtain a homogenate of the animal brain;

- centrifugation of the obtained homogenate of the animal brain;

- purification of the animal brain homogenate from solid components to obtain a supernanant;

- purification of the obtained supernatant from lipid components by extraction to obtain an unclarified protein-peptide complex;

- removal of molecules weighing more than 200,000 Daltons from the obtained unclarified protein-peptide complex to obtain the claimed protein-peptide complex.

At the stage of purification of the obtained supernatant from lipid components, extraction can be carried out using any acceptable extractant used to isolate lipids from animal raw materials, in accordance with the methods specified in [Vostrikova NL, Kuznetsova OA, Kulikovsky A.V. Methodological aspects of extracting lipids from biological matrices // Theory and practice of meat processing. 2018. No. 2].

In this case, the fragmentation of the animal brain tissue with the receipt of fragmented parts of the animal brain tissue is carried out after 1-70 days after the damaging effect on the animal brain tissue, and both chemical and physical effects can be exerted as a damaging effect on the animal brain tissue ...

As animals, mammals, for example pigs, can preferably be used.

Alternatively, the obtained protein-peptide complex is additionally subjected to freezing and subsequent lyophilization.

The claimed protein-peptide complex can be obtained, for example, as follows:

At the first stage, a damaging mechanical effect is exerted on the brain tissue of the animal (pig). In 24 hours after the damaging effect, the animal's brain tissue is fragmented to obtain fragmented parts of the animal's brain tissue, and the fragmented parts of the animal's brain tissue are homogenized at pH 7.3-7.6 to obtain a homogenate of the animal's brain. The resulting homogenate is centrifuged for 25 minutes (10000 g) and is purified from solid components by decantation. The resulting supernatant is purified from lipid components by extraction. Hexane is used (mixed with the supernatant 1 to 3 by volume), the extraction is carried out twice. From the obtained unclarified protein-peptide complex, molecules weighing more than 200,000 Daltons are removed (by passing the complex through a filter with a pore size of 0.48 μm for 2 hours). The resulting preparation is frozen at a temperature below -30 ° C, and then subjected to lyophilization.

Determination of the protein / peptide composition of the obtained protein-peptide complex is carried out by mass spectrometry in a frontal dependent tandem scanning mode on a time-of-flight quadrupole mass spectrometer using the separation of peptides obtained after enzymatic cleavage of protein molecules on a high-performance liquid chromatography system.

The search for and identification of proteins / peptides in the obtained data was carried out using the MASCOT / Andromeda algorithm against the Uniprot 2.2014 database, data validation against a database of decoy random sequences with the maximum permissible threshold value of false positive posik values not exceeding FDR = 1% at a probability of p = 0.05.

Relative quantitative analysis was performed using the iBRQ (intensity-based relative quantification) algorithm with the measurement of the emPAI coefficient, which reflects the repeatability and degeneracy of identified and registered proteotypic peptides for each protein identified in the analysis.

Data on the quantitative content of proteins / peptides in the obtained sample are presented in Table 3:

The solution to this problem also provides a drug for the treatment of cerebrovascular diseases, containing the claimed protein-peptide complex and pharmaceutically acceptable additives, as well as a method for the treatment of cerebrovascular diseases, including the introduction of the patient with the specified drug.

The effectiveness of the claimed protein-peptide complex is illustrated by the following examples.

Example 1. The effectiveness of the claimed protein-peptide complex.

Materials and methods

To determine the effectiveness of the claimed protein-peptide complex, experiments were carried out on male Wistar rats weighing 200-300 g. Hemorrhagic stroke was simulated in animals by the method of A.N. Makarenko (2001) and ischemic stroke - by photothrombosis.

The studied animals were divided into 6 groups. Of these, 2 control groups (with model hemorrhagic and ischemic stroke) and 4 experimental (2 with hemorrhagic and 2 with ischemic stroke). The claimed protein-peptide complex (hereinafter referred to as the drug) was received by 1 group with hemorrhagic and 1 group with ischemic stroke. The comparison groups received a drug for the treatment of ischemic and hemorrhagic stroke, isolated from the brain of pigs subjected to modeling of hemorrhagic stroke, and representing an amino acid-peptide complex, characterized in that it is isolated from the brain of pigs subjected to modeling of hemorrhagic hemispheric stroke after occlusion of the common carotid artery on on the same side (ischemic stroke), obtained in accordance with the text of patent RU 2470650 (hereinafter referred to as analogue). The mortality rate of animals in all groups was assessed.

3 days after the simulation of the model stroke, the surviving animals were injected with samples for 5 days. The control groups received 2 drops of 0.9% NaCl solution intranasally into each nostril. Experimental groups received 2 drops in each nostril of the claimed drug containing 0.2 mg of the drug, and the comparison group received an analogue in the same dosage. According to the Stroke-index McGrow scale as modified by I.V. Gannushkina performed neurological testing of the functions of the surviving animals.

Statistical calculations were performed by the method of analysis of variance with repeated measurements in the program R v.3.4.2. Differences were considered statistically significant at p <0.05

Research results

The following are the results of this study (tables 4, 5):

The discussion of the results

The study of the survival rate of animals after a model stroke showed that the use of the claimed protein-peptide complex significantly (p <0.05) increases the survival rate in comparison with the reference drugs. On each test day presented in Table 5, when using the claimed protein-peptide complex, the neurological deficit was less pronounced compared to the groups where the analogue preparation was used (p <0.05). The neurological deficit that arose in rats after modeling hemorrhagic and ischemic stroke decreased significantly (p <0.05) faster in animals receiving the claimed protein-peptide complex compared with the comparison groups.

Based on the above, it can be concluded that the claimed protein-peptide complex is effective for treating cerebrovascular accidents and restoring lost brain functions, as well as the conclusion that the claimed drug is highly effective in treating both ischemic and hemorrhagic stroke.

Example 2. Study of the effectiveness of the claimed protein-peptide complex.

Purpose of the study

The aim of the study is to compare the effectiveness of the claimed protein-peptide complex, obtained in accordance with two different variants of the method for its production, in models of hemorrhagic and ischemic stroke in vivo.

Materials and methods

Samples of the protein-peptide complex were obtained as follows:

For the first sample of the protein-peptide complex (sample 1): At the first stage, an intravital physical damaging effect was exerted on the pig's brain (infliction of traumatic brain injury using a pistol for stunning cattle Efa cash magnum 9000 s). In 48 hours after the damaging effect, the animal's brain was cleaved, and the fragmented parts of the brain were homogenized at pH 7.4. The resulting homogenate was centrifuged for 25 minutes (10000 g) and was purified from solid components by decantation. The resulting supernatant was purified from lipid components by extraction. Chloroform (mixed with the supernatant 1 to 4 by volume) was used as an extractant; the extraction was carried out three times. Molecules weighing more than 200,000 Daltons were removed from the obtained unclarified protein-peptide complex (by passing the complex through a filter with a pore size of 0.48 μm for 2 h). The resulting preparation was frozen at a temperature of -35 ° C, and then lyophilized.

For the second sample of the protein-peptide complex (sample 2): At the first stage, an intravital chemical damaging effect was exerted on the pig's brain (by inducing oxygen starvation of brain cells caused by vascular occlusion). 45 days after the damaging effect, the animal's brain was cleaved, and the fragmented parts of the brain were homogenized at pH 7.2. The resulting homogenate was centrifuged for 25 minutes (10000 g) and was purified from solid components by decantation. The resulting supernatant was purified from lipid components by extraction. Acetone was used as an extractant (mixed with the supernatant 1 to 2 by volume); the extraction was carried out twice. Molecules weighing more than 200,000 Daltons were removed from the obtained unclarified protein-peptide complex (by passing the complex through a filter with a pore size of 0.48 μm for 2 h). The resulting preparation was frozen at a temperature of -35 ° C, and then lyophilized.

Determination of the protein / peptide composition of the obtained samples of the protein-peptide complex was carried out by mass spectrometry in a frontal dependent tandem scanning mode on a time-of-flight quadrupole mass spectrometer using the separation of peptides obtained after enzymatic cleavage of protein molecules on a high-performance liquid chromatography system.

The search and identification of proteins / peptides in the obtained data was carried out using the MASCOT / Andromeda algorithm against the Uniprot 2.2014 database.

Relative quantitative analysis was performed using the iBRQ (intensity-based relative quantification) algorithm with the measurement of the emPAI coefficient, which reflects the repeatability and degeneracy of identified and registered proteotypic peptides for each protein identified in the analysis.

Data on the quantitative content of proteins / peptides in the obtained sample are presented in table 6:

To determine the effectiveness of the studied samples of the claimed protein-peptide complex, experiments were carried out on male Wistar rats weighing 200-300 g. Hemorrhagic stroke was simulated in animals by the method of A.N. Makarenko (2001) and ischemic stroke by photothrombosis.

The studied animals were divided into 8 groups. Of these, 2 control groups (with model hemorrhagic and ischemic stroke) and 6 experimental (3 with hemorrhagic and 3 with ischemic stroke). Sample 1 received 1 group with hemorrhagic and 1 group with ischemic stroke. Sample 2 was received by group 2 with hemorrhagic and group 2 with ischemic stroke. The comparison groups received a drug for the treatment of ischemic and hemorrhagic stroke, isolated from the brain of pigs subjected to modeling of hemorrhagic stroke, and representing an amino acid-peptide complex, characterized in that it is isolated from the brain of pigs subjected to modeling of hemorrhagic hemispheric stroke after occlusion of the common carotid artery on on the same side (ischemic stroke), obtained in accordance with the text of patent RU 2470650 (hereinafter referred to as analogue). The mortality rate of animals in all groups was assessed.

3 days after the simulation of the model stroke, the surviving animals were injected with samples for 5 days. The control groups received 2 drops of 0.9% NaCl solution intranasally into each nostril. The experimental groups received 2 drops in each nostril of the claimed drug, and the comparison groups received an analogue in the same dosage (0.2 mg). Also, within the framework of the study on the McGrow Stroke-index scale as modified by I.V. Gannushkina performed neurological testing of the functions of the surviving animals.

Statistical calculations were performed by the method of analysis of variance with repeated measurements in the program R v.3.4.2. Differences were considered statistically significant at p <0.05.

Research results

The following are the results of this study (tables 7, 8):

The discussion of the results

The study of the survival rate of animals after a model stroke showed that the claimed protein-peptide complex (samples 1 and 2) significantly (p <0.05) more effectively increases the survival rate of animals in comparison with both the control and the reference drug. The severity of neurological deficit that arose in rats after modeling hemorrhagic and ischemic stroke, in the overwhelming majority of cases (and starting from day 10 of the study - in each case) decreased significantly (p <0.05) faster in animals receiving samples of the claimed protein-peptide complex , in comparison with both the control and the reference drug.

Based on the above, it can be concluded that the claimed protein-peptide complex is effective for treating cerebrovascular accidents and restoring lost brain functions, as well as the conclusion that the claimed drug is highly effective in treating both ischemic and hemorrhagic stroke. It is also worth noting that a significant difference in the composition, and, consequently, in the effectiveness of the studied samples of the claimed protein-peptide complex, obtained in accordance with two different variants of the implementation of the method for its preparation, was not found within the framework of the study.

Claims (3)

1. Protein-peptide complex with neuroprotective and neuroregenerative properties, including a mixture of protein molecules and peptides with a molecular weight of 2500-200000 Daltons, obtained from the brain of a pig subjected to intravital damage to brain tissue by mechanical action and isolated by homogenization of animal brain tissue , purification of the homogenate from solid components to obtain a supernatant, purification of the obtained supernatant from lipid components to obtain an unclarified protein-peptide complex, isolation of a mixture of protein molecules and peptides with a molecular weight of 2500-200000 Daltons from the obtained unclarified protein-peptide complex to obtain a protein-peptide complex ... 2. Protein-peptide complex according to claim 1, characterized in that it contains Complexin-1; Heal shock 70 kDa protein; 14-3-3; Alpha-synuclein, Beta-synuclein; Fatty acid-binding protein, brain; Phosphatidylethanolamine-binding protein 1; Hippocampal cholinergic neurostimulating peptide, Dihydropyrimidinase-related protein 2; Cystatin-B, Tenascin, Cell division cycle 5-like protein, Macrophage migration inhibitory factor. 3. Protein-peptide complex according to claim 1, characterized in that it additionally contains Aspartate aminotransferase, cytoplasmic; Glucoses-phosphate isomerase, Protein DJ-1.